Nanopore-based multiplexed reporter assay system and method

Abstract

A method for parallel (multiplexed) assessment of multiple reporter constructs in living cells is described, in which a population of RTU constructs is introduced into assay cells, cellular RNA is isolated and annealed to a detection oligonucleotide that contains a sequence complementary to the RTU reporter sequence and an adapter sequence conducive for RNA sequencing by a nanopore sequencing device, optionally with amplification of reporter RNA transcripts before detection nucleotide annealing, sequencing annealed reporter RNA or its amplified cDNA using a nanopore sequencing instrument, analyzing the sequencing data to identify and tally processing tags of individual RTUs, and calculating and generating an RTU activity profile as the frequency of counted processing tags. Methods are also described for real-time controlling of quality of multiplexed reporter construct detection, and for assessment of multiple reporter constructs and gene expression in living cells.

1 . A method for the parallel assessment of the activity of multiple reporter constructs in living cells, said method comprising:
 (a) use of a uniform population of reporter transcription units (RTU), each containing an identical reporter sequence;   (b) distinction of each RTU reporter transcript by a processing tag embedded within the reporter sequence, said processing tag either representing a restriction enzyme site variably located within reporter sequences or a barcoding nucleotide sequence, wherein said tags do not interfere with RTU transcription;   (c) introducing the RTU population into assay cells;   (d) isolating cellular RNA, and annealing same to an RTU detection oligonucleotide, which possesses a sequence complementary to the RTU reporter sequence and a facilitating adapter sequence for RNA sequencing by a nanopore sequencing device;   (e) optionally amplifying reporter RNA transcripts before the annealing (d) of said detection oligonucleotide via consecutive reverse transcription and PCR using RTU-specific primers;   (f) sequencing the annealed reporter RNA or its amplified cDNA through a nanopore sequencing device;   (g) analyzing sequencing output to identify and enumerate processing tags associated with individual RTUs; and   (h) calculating and generating an RTU activity profile as the frequency of the counted processing tags.   
     
     
         2 . The method of  claim 1 , wherein said introducing (c) comprises transient or stable transfection, electroporation, or injection of the assay cells. 
     
     
         3 . A method for real-time controlling of quality of multiplexed reporter construct detection in the method of  claim 1 , said real-time controlling method comprising:
 (a) assessing RTU activity profiles calculated and generated in step (h) at two consecutive time points;   (b) calculating similarity of said RTU activity profiles using a Pearson correlation coefficient or Euclidean distance technique; and   (c) stopping the sequencing once the RTU profiles' similarity reaches a predetermined quality threshold.   
     
     
         4 . A method for combined assessment of multiple reporter constructs and gene expression in living cells, said method comprising:
 (a) use of a uniform population of reporter transcription units (RTU) and introduction of said RTU population into assay cells according to steps (a)-(c) defined in  claim 1 ;   (b) isolating cellular mRNA;   (c) annealing one part of said isolated mRNA to an RTU detection oligonucleotide according to steps (d)-(e) defined in  claim 1 ;   (d) annealing the other part of said isolated mRNA to an RNA detection oligonucleotide containing a poly(T) sequence complementary to the 3′ poly(A) RNA tail and an adapter sequence facilitating RNA sequencing by a nanopore sequencing device;   (e) mixing the annealed RNAs and sequencing the mix using a nanopore sequencing device, or   (f) alternatively, sequencing the annealed RNAs separately;   (g) analyzing the sequencing output to identify and enumerate processing tags associated with individual RTUs and calculating and generating an RTU activity profile as the frequency of the counted processing tags; and   (h) analyzing the sequencing output to calculate and generate an output of the expression of endogenous mRNA.   
     
     
         5 . A kit for nanopore-based assessment of multiple reporter constructs, said kit comprising RTU constructs, detection reagents, and software for analyzing the sequencing output and calculating RTU activity profiles and gene expression according to the method of  claim 1 .